1. Field of the Invention
The present invention relates to a process for controlling androgen receptor-regulated mechanisms in mammalian cells under histone H3 at threonine 11- (H3T11-) phosphorylating conditions. Furthermore, the invention relates to a use of inhibitors having specificity for at least one protein kinase C-related kinase (PRK) for controlling androgen receptor-regulated mechanisms in mammalian cells.
2. Discussion of Background Information
Posttranslational modifications of histones such as methylation, acetylation and phoshorylation regulate chromatin structure and gene expression1. Threonine and serine residues are phosphorylated by specific kinases that stay under the control of signaling pathways. A phosphorylation of histone H3 at threonine 11 (H3T11) has not been linked to transcriptional regulation. The protein kinase C-related kinase 1 (PRK1)2 has been shown to phosphorylate H3T11 upon ligand-dependent recruitment to androgen receptor (AR) target genes. H3T11 phosphorylation is an early event that precedes demethylation of mono-, di-, and trimethyl histone H3 at lysine 9 by JMJD2C and lysine specific demethylase 1 (LSD1). PRK1 is pivotal to AR function, since PRK1 knockdown by RNAi or PRK1 inhibition by treatment with Ro318220 impedes AR-dependent gene expression. Blocking PRK1 function abrogates androgen-induced phosphorylation of H3T11, but also blocks, in consequence, demethylation of mono-, di-, and trimethyl H3K9 as well as acetylation of histone H3 at lysines 9 and 14 (H3K9 and H3K14). Moreover, the presence of serine 5-phosphorylated RNA polymerrase II is no longer observed at AR target promoters. Thus, phosphorylation of H3T11 by PRK1 establishes a novel epigenetic mark for transcriptional activation, identifying PRK1 as a gatekeeper of AR-regulated gene expression. This pathway is of utmost importance since knockdown of PRK1 in prostate cancer cells inhibits androgen-induced transcriptional activation and tumor cell proliferation. Thus, our data suggest that specific gene regulation requires the assembly and coordinate action of kinases and demethylases. Furthermore, regulation of PRK1 activity alone or in combination with LSD1 and JMJD2C might be a promising therapeutic strategy to control AR activity in prostate cancer. Importantly, high PRK1 levels positively correlate with high Gleason scores of prostate carcinomas, allowing the present invention to be used in scoring prostate carcinomas.
The N-terminal tails of histones are subject to a plethora of posttranslational modifications such as acetylation, phosphorylation, and methylation by specific chromatin-modifying enzymes1. During gene expression, these modifications influence chromatin structure to facilitate the assembly of the RNA polymerase II transcription machinery1, 3. Androgen receptor (AR)-dependent gene expression is characterized by epigenetic changes such as removal of repressive methyl marks from lysine 9 of histone H3 (H3K9)4, 5 and acetylation of lysines 9 and 14 of histone H3 (H3K9/K14)6. However, little is known about the upstream regulators that govern these epigenetic modifications. Since protein kinase C-related kinase 1 (PRK1) controls AR-dependent gene expression2, we asked whether PRK1 signaling regulates epigenetic events at AR target genes.
Hence, it was an object of the present invention to identify further modulators of the AR-regulated gene expression and/or androgen-induced cell proliferation, particularly in mammalian cells.
Moreover, it was an object of the present invention to provide further modulators of histone modification, in particular of histone phosphorylation, methylation and acetylation.
Another object of the invention was to provide processes for controlling at least one androgen receptor-regulated mechanism in mammalian cells and for controlling the transcriptional AR activation induced by different routes.
A further object of the invention was to provide a new process for the prevention and/or treatment of prostate cancer.
Another object of the invention was to provide for the use of one or more than one inhibitor for the medicament manufacture, particularly for manufacturing a medicament for preventing and/or treating cancer, particularly prostate cancer.
Furthermore, it was an object of the invention to provide a means for scoring prostate cancers, particularly an assay system.
Finally, it was an object of the invention to provide an assay system for inhibitors having specificity for at least one PRK capable of blocking AR-induced prostate carcinoma cell proliferation.